Bone seeking technetium 99m complex

ABSTRACT

A technetium-99m-stannous-phosphate complex in which the phosphate moiety includes pyrophosphate and in which no more than 5 to 15% or 20% by weight of such phosphate moiety is a linear polyphosphate of molecular weight greater than pyrophosphate, a method of making the same, a method of using the same by in vivo intravenous administration to a mammal of the sterile, nonpyrogenic complex followed by radioassay scanning or imaging the skeletal structure, and a kit made up of a stannous-phosphate complex in which the phosphate moiety is pyrophosphate and in which no more than 5 to 15% or 20% by weight of such phosphate moiety is a linear polyphosphate of molecular weight greater than pyrophosphate.

This is a division of application Ser. No. 410,086 filed Oct. 27, 1973, now U.S. Pat. No. 4,016,249 which is a division of application Ser. No. 288,577, now U.S. Pat. No. 3,851,044, filed Sept. 13, 1972 on the same day as application Ser. No. 288,683, now U.S. Pat. No. 3,852,414.

BACKGROUND OF THE INVENTION

It has been known for some time that phosphates, including long chain linear polyphosphates, when introduced into the blood stream of mammals will selectively seek out and collect in the bone or skeletal structure. Pro. Soc. Exp. Biol Med. Volume 100, pages 53-55 (1959); Journal of Labelled Compounds, April-June 1970, Vol. VI, No. 2, pages 166-173; Journal of Nuclear Medicine, Vol. 11, No. 6, pages 380-381, 1970; Journal of Nuclear Medicine, Vol. 1, No. 1, January 1960, pages 1-13. In these cases a phosphorous atom or atoms of the phosphate are radioactive, i.e. ³² P.

It has also been known for some time that technetium-99m (^(99m) Tc) is a preferred radionuclide for radioactively scanning organs because of its short half life and because it radiates gamma rays which can be easily measured, compared, for example, to beta rays. See Radiology, Vol. 99, April 1971, pages 192-196.

It has also been known for some time to use divalent stannous tin (Sn⁺⁺) in the form of stannous chloride, or divalent iron (Fe⁺⁺) or reduced zirconium to bind radioactive ^(99m) Tc to carriers, such as chelating agents, red blood cells, albumin and other proteins, which selectively seek out certain organs of the body, in order to carry the ^(99m) Tc with them to such organs of the body where it is concentrated, whereby such organ can be radioactively scanned or imaged for diagnostic or other purposes, e.g. radioactive treatment of a pathological condition. See Journal of Nuclear Medicine, Vol. 11, No. 12, 1970, page 761; Journal of Nuclear Medicine, Vol. 12, No. 1, 1971, pages 22-24; Journal of Nuclear Medicine, Vol. 13, No. 2, 1972, pages 180-181; Journal of Nuclear Medicine, Vol. 12, No. 5, May 1971, pages 204-211; Radiology, Vol. 102, January 1972, pages 185-196; Journal of Nuclear Medicine, Vol. 13, No. 1, 1972, pages 58-65.

Also, it has been suggested to label a stannous compound with ^(99m) Tc for radioactively imaging bone marrow, Journal of Nuclear Medicine, Vol. 11, 1970, pages 365-366.

It has also been known for some time that the stannous ion Sn⁺⁺ forms soluble complexes with long chain polyphosphates, Journal Inorganic Nuc. Chem., Vol. 28, 1966, pages 493-502.

It has been suggested to employ the aforesaid ^(99m) Tc for radioactively scanning the skeletal bone structure of mammals by complexing or binding it to tripolyphosphate carrier by use of the aforesaid stannous ion as a binding agent in order for such phosphate to selectively carry the ^(99m) Tc to, and concentrate it in, the skeletal bone structure upon in vivo intravenous administration for subsequent radioactive scanning or imaging the skeletal structure. Radiology, Vol. 99, April 1971, pages 192-196. The use of ^(99m) Tc in this manner is alleged to have certain advantages over the use of strontium, e.g. ⁸⁵ Sr, as the radioactive label which has been used for radioactive bone scanning in the past. These advantages are those which are inherent in ^(99m) Tc, i.e. short half life and pure, near optimal energy gamma rays. However, the bone uptake (the percent of the total dosage which becomes concentrated in the skeletal structure within a certain time after in vivo intravenous administration) of such ^(99m) Tc-containing complex and the ratios of such bone uptake to uptake of the ^(99m) Tc by the other organs of the body (the higher these ratios the better), i.e. radioactive contrast, are not nearly as high as with radioactive strontium.

STATEMENT OF THE INVENTION

It has been discovered that if the phosphate moiety of the ^(99m) Tc-stannous-phosphate complex, which has been suggested in the aforesaid Radiology publication, comprises pyrophosphate (P₂ O₇ ⁻⁴) (which is a linear polyphosphate moiety of molecular weight less than 300), the bone uptake, bone/blood ratio, bone/liver ratio, bone/G.I. ratio and bone/kidneys ratio are substantially increased.

It has also been discovered that optimum results are achieved if such phosphate moiety contains no more than about 15 to 20 or 25%, preferably no more than 5 to 10% and more preferably no more than 5% (less than 5% is the most preferred), by weight of linear or branched chain polyphosphate (formula P_(n) O_(3n+1) ⁻(n+2)) of molecular weight greater than that of pyrophosphate.

Maximum bone/liver ratios are achieved when the ^(99m) Tc-Sn⁺⁺ - pyrophosphate complex is administered to the mammal in relatively small dosages of substantially less than 20 or 25, preferably substantially less than 8 or 10 and still more preferably less than 5 or 6 (between 0.01 or 0.10 and 3 and even less provide excellent results), milligrams pyrophosphate moiety per kilogram of body weight of the mammal.

The term "phosphate moiety" as used herein refers to the phosphorus and oxygen atoms only of the phosphate.

The presence of polyphosphates of formula P_(n) O_(3n+1) ⁻(n+2) and molecular weight greater than that of pyrophosphate seems to reduce bone take-up and the aforesaid ratios, as compared to complexes without such higher molecular weight polyphosphates. However, as aforesaid, some of such higher molecular weight polyphosphates can be tolerated, preferably not more than about 15% to 20% or 25%, more preferably no more than 5% to 10% and still more preferably not more than 5% (less than 5% is the most preferred), by weight of the total phosphate moiety.

Where the pyrophosphate does not constitute 100% of the phosphate moiety of the ^(99m) Tc-Sn⁺⁺ -phosphate complex, the rest of the phosphate moiety is preferably a ring phosphate of formula P_(n) O_(3n) ^(-n) (with n preferably being 3 which is trimetaphosphate) and/or ortho phosphate, and preferably a ring phosphate only, although the aforesaid limited amounts of higher molecular weight linear polyphosphates can be tolerated.

The complex is made from a water soluble alkali metal (preferably sodium) or ammonium salt or acid salt of the pyrophosphate, e.g. sodium pyrophosphate.

Preferably, the sodium pyrophosphate is admixed with a stannous salt, e.g. SnCl₂ (the stannous salts of other acids which are pharmaceutically acceptable, i.e. safely intravenously administered, can be used) to form the stannous-pyrophosphate complex, the pH of which is adjusted to 3-8, preferably 5-8, by a pharmaceutically acceptable acid, such as HCl, or base, such as NaOH or Na₂ CO₃ or NaHCO₃, followed by admixing with the stannous-pyrophosphate complex, an aqueous saline solution of radioactive sodium pertechnetate (^(99m) Tc) to form the ^(99m) Tc-stannous-pyrophosphate complex at the time it is desired to intravenously administer the ^(99m) Tc complex. The stannous-pyrophosphate complex may be sealed in a sterile, non-pyrogenic container or vial as a solution or a lyophilized solid and shipped as a kit with the freshly generated sterile and non-pyrogenic ^(99m) Tc being added aseptically at the situs just prior to use.

DETAILED DESCRIPTION OF INVENTION (Including Examples)

The following compositions were prepared:

                  TABLE I                                                          ______________________________________                                         Sample                                                                         No.   Description                                                              ______________________________________                                         1     A commercial sodium polyphosphate sold by FMC                                  Corporation under the trade name FMC Glass H                                   (average chain length of 21 and average M.W. about                             2100).                                                                   1-1   A first high molecular weight fraction of the FMC                              Glass H of Sample 1 obtained by fractionating an                               aqueous solution of Sample 1 with acetone according                            to the technique described in Van Wazer, Phosphorous                           And Its Compounds, Interscience Publishers, Inc.                               1961 (pages 744-747) to precipitate out of the                                 aqueous solution of the FMC Glass H, as an oil, the                            highest molecular weight fraction of polyphosphates                            (composition given in TABLE 2).                                          1-2   A second acetone fraction of FMC Glass H achieved by                           adding more acetone to precipitate out of the                                  remaining supernatant of 1-1, as an oil, the next                              higher molecular weight polyphosphates (composition                            given in TABLE 2). The acetone decreases the                                   solubility of the polyphosphates in the water; the                             higher the molecular weight of the polyphosphate the                           less soluble it is so that the highest molecular                               weights are forced out of solution first.                                1-3   A third acetone fraction of FMC Glass H containing                             the next higher molecular weight polyphosphates is                             precipitated out of the remaining supernatant                                  solution of 1-2, as an oil, upon addition of further                           amounts of acetone (composition given in TABLE 2).                       1-4   A fourth acetone fraction of FMC Glass H containing                            the next higher molecular weight polyphosphates                                (composition given in TABLE 2) is precipitated out                             of the supernatant solution of 1-3, as an oil, by                              adding more acetone.                                                     1-5   A fifth acetone fraction of the FMC Glass H                                    (containing the next higher molecular weight poly-                             phosphates) (composition given in TABLE 2) is pre-                             cipitated out of the remaining supernatant solution                            of 1-4, as an oil, by adding more acetone.                               1-6   A sixth acetone fraction of FMC Glass H (composition                           given in TABLE 2) is precipitated out of the remain-                           ing supernatant solution of 1-5, as a solid pre-                               cipitate of the next higher molecular weight poly-                             phsophates by adding more acetone.                                       1-7   A seventh acetone fraction of FMC Glass H (composi-                            tion given in TABLE 2) is precipitated out of the                              remaining supernatant solution of 1-6, as a solid                              precipitate of the next higher molecular weight                                polyphosphates by adding more acetone.                                   1-8   The residue fraction in the supernatant liquid left                            after removal of the 1-7 fraction (composition given                           in TABLE 2) is recovered by evaporating off the                                supernatant liquid.                                                      2     An end acetone fraction of Sample 1 after 90% by                               weight had been previously fractionated off and                                leaving after removal of such and fraction 3% by                               weight in the supernatant (Composition given in                                TABLE 2).                                                                4     A mixture of 86% sodium trimetaphosphate (Na.sub.3 P.sub.3 O.sub.9),           1                                                                              3% sodium orthophosphate (Na.sub.3 PO.sub.4) (molecular weight                 of phosphate moiety - 95) and 10% sodium pyro-                                 phosphate (Na.sub.4 P.sub.2 O.sub.7) (linear polyphosphate -                   molecular                                                                      weight of phosphate moiety - 174) obtained by                                  acetone fractionation of sodium trimetaphosphate                               obtained from Monsanto. Sodium trimetaphosphate, -  as aforesaid,              is one of a plurality of cyclic                                                phosphates having the general formula P.sub.n O.sub.3n.sup.-n.                 Sodium orthophosphate is a phosphate monomer.                                  Sodium pyrophosphate is a dipolyphosphate.                               5     An acetone end fraction of a food grade polyphosphate                          sold by FMC under the name FMC FG (composition given                           in TABLE 2).                                                             6     A commercial cyclic trimetaphosphate sold by                                   Stauffer Chemical. (composition given in TABLE 2).                       7     Sodium orthophosphate.                                                   8     Sodium pyrophosphate.                                                    9     Sodium tripolyphosphate                                                  10    Sodium tetrapolyphosphate - Na.sub.6 P.sub.4 O.sub.13 - a poly-                phosphate - phosphate moiety having a M. W. of 348.                            It, together with the pyrophosphate and tripoly-                               phosphate, fall in the class of linear chain poly-                             phosphates having the general formula P.sub.n O.sub.3n + 1.sup.-(n             + 2).                                                                    ______________________________________                                    

An aqueous solution of each of the phosphate composition samples 1 through 10 (40 mg. phosphate/1 ml. solution) were made with distilled water in which the dissolved oxygen content was reduced in a conventional manner by bubbling through such water gaseous nitrogen for a period of two hours. The water and phosphates were mixed to form the solutions in a nitrogen atmosphere and in a nitrogen flushed container. The reason for this is to reduce oxidation of the divalent Sn⁺⁺ to be subsequently admixed with each solution sample. However, it is not essential (but highly preferred) to use nitrogen-treated water or a nitrogen atmosphere or a nitrogen-flushed container. Other known pharmaceutically acceptable conditions, which will inhibit oxidation of the Sn⁺⁺ upon subsequent mixing thereof with the phosphate solution, can be used, including the use of conventional pharmaceutically acceptable reducing agents and anti-oxidants in the products used.

Each of these solutions, samples 1 through 10, in an amount equal to 100 ml, was mixed with 0.16g of solid SnCl₂. 2H₂ O under a nitrogen atmosphere. The SnCl₂. 2H₂ O was made by adding to 84.5 mg. of metallic tin, sufficient concentrated HCl with mixing until all the tin has dissolved followed by removing excess acid and water by lyophilization (this operation also being carried out in a vacuum or in a nitrogen atmosphere and in a nitrogen flushed container to prevent oxidation of stannous to stannic). Antioxidants, which can be administered intravenously, may also be used. A stannous (Sn⁺⁺)-phosphate complex or mixture of some kind was formed in each case, the phosphate moiety of each sample corresponding to the phosphate moieties of the phosphates set forth in TABLE 2.

Sufficient aqueous solution 3N sodium hydroxide (sodium carbonate or bicarbonate can also be used), in the case of samples 1 through 7 and 9 and 10, and 3N HCl, in the case of sample 8, is then added to each sample to give a pH of 5.0 to achieve a pH suitable for subsequent intravenous in vivo administration into the body of a mammal, in this case adult mice. The pH adjustment is preferably done under a nitrogen atmosphere also.

After thorough mixing, the solutions are sterilized by passing them through a Millipore biological filter of 0.22 micron pore size under a nitrogen atmosphere. Thereafter milliliter portions of each of the sterile solutions are poured into individual sterile and non-pyrogenic storage glass vials under a nitrogen atmosphere.

In the case of each sample, vials are lyophilized by conventional freeze drying equipment under aseptic conditions to remove water. This provides a solid stannous-phosphate complex which aids in shipping and storage and which is more stable than the complex in solution.

Each vial contains 1.35 mg. SnCl₂ and 40 mg. of the phosphate.

The vials can be sealed and stored until needed subsequently to form the technetium-99m-stannous-phosphate complex at the use situs.

To prepare the technetium-99m complex, 3 to 7 (5) ml. of fresh sodium pertechnetate, removed as a sterile non-pyrogenic eluate from a sterile NEN ^(99m) Tc Generator (any other source of pharmaceutically acceptable ^(99m) Tc can be used, including ^(99m) Tc generators manufactured by others than NEN), in a 0.9% saline solution is aseptically added to each vial containing the sterile and non-pyrogenic stannous-phosphate complex and the vial is swirled until a solution is obtained. In each case a technetium-99m-stannous-phosphate complex or mixture of some kind is formed in aqueous solution (8 mg. phosphate per ml solution when 5 ml of pertechnetate is used), the phosphate moiety of which corresponds to the phosphate moieties of the phosphate compounds of each sample set forth in TABLE 2.

Aseptic techniques and sterile, non-pyrogenic ingredients and containers were used at all steps, such procedures being standard to those skilled in the art.

An eleventh sample was prepared in the manner set forth above by diluting sample 8 to a concentration of 1 mg of phosphate per ml of solution. This was labeled Sample 11.

Each of the technetium-99m-stannous-phosphate complex-containing solutions is aseptically intravenously injected in vivo into a vein in the tail of adult mice (average weight 0.040 kgs) in an amount equal to between 1 and 3 mCi and a volume of 0.12 ml (8 mg. of phosphate per ml solution in samples 1 through 10 and 1 mg. phosphate per ml. solution in sample 11). Also sample 8 was injected in the same manner except that a volume of 0.015 ml was injected instead of 0.12 ml to reduce the dosage of the complex by a factor of 8. This was labeled Sample 12.

Three hours after intravenous administration, some of the mice to which each sample was administered were sacrificed and the various organs of their bodies (skeletal, liver, G.I., blood, kidneys) were counted by conventional gamma ray counting techniques to determine uptake of ^(99m) Tc by each organ and thereby determine contrast of bone uptake as compared to uptake by the other organs. As aforesaid, it is not only important to have a high bone uptake (based on total technetium-99m-dosage) but it is also important that the ratio of uptake by the bone to uptake by the other organs be high.

The results are set forth in TABLE 2 below, in which the uptakes (the bone uptake figures represent the average bone uptake for the skeletal system) are in terms of percent of the total technetium-99m activity injected (corrected for radioactive decay) which has collected in the various organs indicated three hours after in vivo intravenous injection, in which the ratio amounts are computed from the uptake amounts, in which "Percent Having Phosphate Moiety M. W. Less Than 300" refers to weight percent of the phosphate moiety based on the total phosphate moiety of the sample identified in the first horizontal column, in which the percents referred to under Phosphate Composition are weight percents of the whole phosphate moiety of the sample (as aforesaid, phosphate moiety as used herein is limited to that part of the compound or complex made up of phosphate phosphorus and oxygen atoms), in which Ortho P1 refers to the phosphate moiety of sodium orthophosphate, Pyro P2 refers to the phosphate moiety of sodium pyrophosphate, Tripoly P3 refers to the phosphate moiety of sodium tripolyphosphate, Tetrapoly P4 refers to the phosphate moiety of sodium tetrapolyphosphate, Trimeta R3 refers to the phosphate moiety of sodium trimetaphosphate, Tetrameta R4 refers to the phosphate moiety of sodium tetrametaphosphate, both trimeta and tetrametaphosphates falling within the class of cyclic or ring phosphates having the formula P₃ O_(3n) ^(-n), in which "Pentapoly And Longer Linear Chains" refers to the phosphate moiety of sodium pentapolyphosphate and longer linear (linear as used herein includes straight and branched linear phosphate chains) polyphosphates of formula P_(n) O_(3n+) 1⁻(n+2), in which "Average M.W." refers to the average molecular weight of the phosphate moiety of the sample and in which "Fraction In Raw Stock" with reference to samples 1-1, 1-2, 1-3, 1-4, 1-6, 1-7 and 1-8 refer to the normalized percent by weight of each of these samples in sample 1, which is the raw stock which is fractionated.

                                      TABLE 2                                      __________________________________________________________________________               FMC                                                                  Phosphate Sample                                                                         1  1-1                                                                               1-2                                                                               1-3                                                                               1-4                                                                               1-6                                                                               1-7                                                                               1-8 2  4 5  6  7  8  9  10                                                                               11 12                 __________________________________________________________________________     __________________________________________________________________________     Uptake                                                                         Blood     1.7                                                                               4.0                                                                               4.2                                                                               4.6                                                                               2.9                                                                               3.4                                                                               1.3                                                                               1.3 1.6                                                                               1.3                                                                              0.65                                                                              1.0                                                                               4.5                                                                               1.2                                                                               1.8                                                                               3.3                                                                              1.4                                                                               1.1                Liver     23 2.7                                                                               2.7                                                                               2.8                                                                               2.2                                                                               6.8                                                                               0.9                                                                               2.0 1.3                                                                               1.3                                                                              0.52                                                                              0.85                                                                              5.9                                                                               7.5                                                                               1.3                                                                               4.7                                                                              1.2                                                                               0.8                G.I.      4.9                                                                               8.5                                                                               5.5                                                                               7.5                                                                               4.2                                                                               4.8                                                                               1.8                                                                               1.5 2.2                                                                               1.9                                                                              0.75                                                                              1.9                                                                               1.8                                                                               1.7                                                                               2.2                                                                               4.0                                                                              1.8                                                                               2.5                Kidneys   3.6                                                                               3.7                                                                               3.1                                                                               3.2                                                                               1.7                                                                               2.6                                                                               0.9                                                                               0.9 1.1                                                                               1.5                                                                              0.88                                                                              0.82                                                                              1.1                                                                               1.3                                                                               1.5                                                                               3.1                                                                              1.3                                                                               3.0                Av. Bones 17 35 31 32 31 35 45 65  53 70                                                                               70 57 20 56 45 29                                                                               82 65                 Ratios                                                                         Bone/Blood                                                                               5  4  4  4  5  5  18 29  17 30                                                                               58 32 2  34 13 5 35 24                  /Liver   0.5                                                                               7  6  7  8  3  29 19  24 32                                                                               78 38 2  5  20 3 47 36                  /G.I.    5  6  8  7  11 11 37 63  36 55                                                                               140                                                                               55 17 49 31 11                                                                               81 32                  /Kidneys 1  1  1  1  3  2  9  12  8  9 13 12 3  9  5  1 12 6                  Percent Having                                                                 Phosphate Moiety                                                               M.W. Less 15%                                                                               3% 3% 5% 13%                                                                               17%                                                                               90%                                                                               100%                                                                               55%                                                                               99                                                                               98 98 100                                                                               100                                                                               100                                                                               10                                                                               100                                                                               100                Than 300                                                                       Phosphate                                                                      Composition                                                                     Ortho P.sup.1 (%)                                                                       3  1.0                                                                               1  2  5  5  10 50  5  3 15 4.0                                                                               100                                                                               0  0  0 0  0                   Pyro P.sup.2 (%)                                                                        2  0.5                                                                               0.5                                                                               1  2  1  15 10  10 10                                                                               5.0                                                                               8.0                                                                               0  100                                                                               0  0 100                                                                               100                Tripoly P.sup.3 (%)                                                                      2  0.5                                                                               0.5                                                                               1  2  1  5         0 0  0  0  0  100                                                                               0 0  0                  Tetra Poly P.sup.4 (%)                                                                   2  0.5                                                                               0.5                                                                               1  3  10 5         1 0  1.0                                                                               0  0  0  90                                                                               0  0                  Trimeta R.sup.3 (%)                                                                      8  0.5                                                                               0.5                                                                               1  4  10 60 40  40 86                                                                               78 86 0  0  0  10                                                                               0  0                  Cyclic Tetra                                                                             4  0.5                                                                               0.5                                                                               1  4  50 5      40   0  0  0  0  0  0 0  0                   Meta R.sup.4 (%)                                                              Penta Poly P.sup.5 &                                                                     79 96.5                                                                              96.5                                                                              93 80 23 0  0   5    2  1  0  0  0  0 0  0                   Longer Linear                                                                  Chains                                                                        Average M.W.                                                                             2100                                                                              3900                                                                              2700                                                                              1800                                                                              1200                                                     Fraction in                                                                              100                                                                               3.5                                                                               61 16.2                                                                              7.5                                                                               5.0                                                                               6.1                                                                               0.7                                              Raw Stock                                                                     __________________________________________________________________________

Conventional gamma counting techniques for measuring technetium 99m take-up in the organs are conventional gamma ray-excitable scintillation counters for radioassaying multiple samples of the organs of the sacrificed mice.

Also, conventional scanning by radioactive imaging using a gamma ray-excited scintillation or gamma camera and a dual crystal rectilinear scanner was used in vivo. In vivo scintiphotos of the total body using the Anger camera were obtained as well as rectilinear total body scans.

The figures given in TABLE 2 are average figures achieved by the aforesaid conventional counting techniques, each sample having been intravenously administered to mice followed by radioactive counting.

Following intravenous administration, the ^(99m) Tc-Sn⁺⁺ - pyrophosphate complexes of the present invention are rapidly cleared from the blood by deposition in bone and excretion into urine. Thus, the technetium-99m-stannous-pyrophosphate complexes are metabolizable. The deposition of the ^(99m) Tc-stannous-pyrophosphate complexes of the invention appears to be primarily a function of the bone blood flow as well as being related to the efficiency of the bone in extracting the complex from the blood which perfuses the bones.

It was observed that the deposition of the ^(99m) Tc in the skeleton is bilaterally symmetrical with increased accumulations being present in the axial skeleton as compared to the appendicular skeleton. There is also increased deposition in the distal aspect of long bones.

Localized areas of abnormal accumulation of the radiopharmaceutical may be seen in primary malignancies of the bone, metastatic malignancies of the bone, acute or chronic osteomyelitis, arthritides, recent fractures, areas of ectopic calcification, Paget's disease, regional migratory osteoporosis, areas of aseptic necrosis and in general any pathological situation involving bone in which there is increased osteogenic activity or localized increased osseous blood perfusion.

The acute toxicity level in mice (LD_(50/30)) for Sample No. 2 has been determined to be 150 mg/Kg body weight and for Sample 6 it is 800 mg/Kg and for Sample 8 it is 70 mg/Kg. Subacute toxicity studies in mice of Sample 2 have shown no signs of toxicity after 15 daily injections at dose levels as high as 63 mg/Kg body weight/day. A similar subacute study in dogs indicates no signs of toxicity at a dose level of 3.6 mg/Kg body weight/day.

It was found that samples 4 and 6 were only one fourth as toxic to mice as sample 2 and one-eighth as toxic to mice as sample 1.

The complexes of the invention have been used successfully as a skeletal imaging or scanning agent to visualize areas of altered blood flow to the bone and altered osteogenic activity, including suspected bone lesions not shown on X-ray, bone survey performed as part of a work-up in patients with known or suspected malignancy, to follow the response of metastatic or primary bone lesions to radiation therapy, metabolic bone disease, to diagnose arthritis and osteomyelitis, and to diagnose and determine healing rate of bone fractures.

Seven clinical studies by seven doctors on humans using sample 2 and involving 91 patients showed no adverse reactions and were deemed to be highly successful and clinically useful for skeletal diagnostic purposes in the case of 90 patients.

The technetium-99m (^(99m) Tc) labeling reactions involved in preparing the ^(99m) Tc-stannous-phosphate complexes of the invention depend on maintaining the tin in the reduced or stannous (Sn⁺²) state. Oxidants present in the pretechnetate supply may adversely affect quality.

The radioactive dosage of the ^(99m) Tc complex of the invention may vary from 1 to 25 mCi (millicuries) but preferably is from 10 to 15 mCi. The dosage should preferably be substantially less than 20 or 25, preferably less than 8 or 10 and more preferably less than 5 to 6 mg. of pyrophosphate moiety per kilogram of body weight of the mammal since greater pyrophosphate dosages than this reduces the bone-liver ratio too much. Note for example the low bone-liver ratio in sample 8 where the dosage was 25 mg. pyrophosphate per Kg body weight compared to the bone-liver ratio of sample 11 where the dosage was 3.1 mg/Kg body weight.

Only trace amounts of pyrophosphate moiety in the dosage, e.g. as low as 0.001, more preferably 0.01, mg/Kg body weight, gives good bone take-up and bone-to-other organ take-up ratios.

The dosage of pyrophosphate can be kept small either by use of more dilute dosage solutions of the pure pyrophosphate, as in sample 11 or by administering smaller doses of a more concentrated complex solution, the phosphate moiety of which contains a high concentration of pyrophosphate or by more concentrated solutions of phosphate containing, in addition to the pyrophosphate, ring phosphate and/or orthophosphate which effectively dilute the pyrophosphate concentration of the dose.

It is preferred to use a ^(99m) Tc-Sn⁺⁺ -- phosphate solution containing between 0.1 and 40, more preferably between 0.5 and 4 or 5 mgs of pyrophosphate moiety per ml of solution.

An advantage of a complex containing a relatively large amount of ring phosphate and a smaller amount of pyrophosphate is that the ring phosphate in addition to providing excellent bone up-take and bone-to-other-organ ratios is less toxic than pyrophosphate, although pyrophosphate, alone, is still not unduly toxic.

Scanning may be commenced as early as one hour after intravenous administration and may be as long after injection as clinically useful amounts of ^(99m) Tc remain in the organ.

Another manner of making the complex of the invention is to weigh 4 mg. of SnCl₂. 2H₂ O and 100 mg. of sodium pyrophosphate into a flask (the flask is sterile and non-pyrogenic and is flushed with nitrogen before weighing and is kept under nitrogen during this step and for the next step). Add, under aseptic conditions, 12 ml of sterile, non-pyrogenic sodium pertechnetate in 0.9% saline solution. Shake the mixture until a solution is obtained followed by intravenous injection (preferably the pH of the mixture is aseptically adjusted to pH 3-8 before intravenous injection).

Also, the sterile stannous chloride can first be aseptically mixed with the sterile ^(99m) Tc saline solution to form a ^(99m) Tc-stannous complex, followed by adding the sterile sodium pyrophosphate under aseptic conditions to form the ^(99m) Tc-stannous-pyrophosphate, adjusting the pH to 3-8, followed by intravenous injection.

It can be seen from TABLE 2 that a ^(99m) Tc-stannous-phosphate complex in which the phosphate moiety comprises pyrophosphate and in which such moiety contains no more than 25% by weight of linear polyphosphate of molecular weight greater than that of pyrophosphate (samples 1-7, 1-8, 2, 4, 5 and 6, 8, 11 and 12) gives surprising higher bone uptake and ratio of bone uptake to other organs, as compared to orthophosphate and other polyphosphates, e.g. tripolyphosphate, tetrapolyphosphate and longer chain polyphosphates (see samples 1, 1-2 to 1-6, 7, 9 and 10).

It can also be seen by comparing samples 8, 11 and 12 in TABLE 2 that the bone to liver ratio is substantially increased by reducing the amount of pyrophosphate in the dosage administered to the mammal.

The pyrophosphate moiety of the ^(99m) Tc-stannous-phosphate complex may be from 1 or 2% or even less up to 100% by weight of the total phosphate moiety. Preferably, the pyrophosphate moiety consists of 5 or 10% or more of the total phosphate moiety, more preferably 50% or 60% or more and most preferably between 90 and 100%.

Although the stannous (Sn⁺⁺) ion is by far preferred, the divalent ferrous (Fe⁺⁺) ion in the form of ferrous ascorbate, and reduced zirconium can also be used but without as good results. All these metals can exist in a plurality of redox states.

The phosphate may be added to the solid SnCl₂ as an aqueous solution, or it may be added to a solution of the SnCl₂ to form the Sn⁺⁺ -phosphate complex followed by adding the ^(99m) Tc solution.

Very little Sn⁺⁺ need be used to form the complex of the invention, e.g. less than 7 or 10% of the phosphate based on molecular weights.

The weight ratio of Sn⁺⁺ ion to the pyrophosphate moiety may vary over a wide range, i.e. from 10⁻³ to 0.50, preferably 0.01 to 0.4. It is preferred that the molecular ratio of Sn⁺⁺ to pyrophosphate moiety not exceed 2/1. The maximum ratio is dictated by the amount beyond which the precipitation of Sn⁺⁺ occurs. The minimum amount required is that amount necessary to bind a sufficient amount of ^(99m) Tc to the pyrophosphate to achieve good bone uptake and contrast. This can be determined by routine experiment.

The pH of the stannous-phosphate complex may be between 3 and 8.

The water used for making the complexes of the invention is distilled and is at an elevated temperature of 200° F during removal of dissolved oxygen and reduction of oxidants by bubbling the nitrogen gas therethrough.

The maximum amount of ^(99m) Tc is that beyond the capacity of the Sn⁺⁺ -pyrophosphate complex to bind the ^(99m) Tc. This can be determined by routine thin layer radiochromatography to determine the percent of free or unbound ^(99m) Tc in the complex. The minimum amount is dictated by that amount below which there is an insufficient amount to give good scanning of bone uptake and contrast, which also can be determined by routine experiment. Generally, the amount of ^(99m) Tc added to the Sn⁺⁺ pyrophosphate complex should be sufficient to achieve the counting rate desired by the doctor or laboratory personnel for the volume to be injected; oridinarily, as aforesaid, the activity dosage varies from 5 to 25 millicuries.

Although sodium pyrophosphates are preferred, any alkali metal, such as potassium and lithium, or ammonium can be used as the cation so long as it is pharmaceutically acceptable so that it can be safely administered intravenously. Also the acid pyrophosphates of such cations can be used.

Although in the examples given above saline water was used as the vehicle, any other vehicle which is pharmaceutically acceptable for intravenous administration can be used.

It is not intended that the invention be limited to any theory which may have been given above or to the specific examples set forth above but only by the claims appended hereto and their equivalents. 

We claim:
 1. A composition for forming a complex with technetium-99m for radioactive scanning, comprising a stannous-phosphate complex, the phosphate moiety of which comprises pyrophosphate, said phosphate moiety containing no more than 25% of linear polyphosphates of formulation P_(n) O_(3n+1) ⁻(n+2) having a polyphosphate moiety of molecular weight greater than pyrophosphate, the weight ratio of stannous to pyrophosphate moiety ranging from 10⁻³ to 0.50.
 2. A composition according to claim 1, in which at least 5% by weight of the phosphate moiety of said complex is pyrophosphate.
 3. A composition according to claim 1, said phosphate moiety being substantially free from said linear polyphosphates.
 4. A composition according to claim 2, substantially 100% by weight of said phosphate moiety being said pyrophosphate.
 5. A composition to claim 1, at least the major portion of any phosphate in said phosphate moiety other than pyrophosphate being selected from the group consisting of a ring phosphate of formula P_(n) O_(3n) ^(-n), orthophosphate and combinations thereof.
 6. A composition according to claim 5, where n is equal to
 3. 7. A composition according to claim 6, at least 5% by weight of said phosphate moiety being said pyrophosphate and said phosphate moiety containing no more than 10% by weight of said linear polyphosphate of molecular weight greater than pyrophosphate.
 8. A composition according to claim 1, said phosphate moiety consisting of said pyrophosphate, a ring phosphate of formula P_(n) O_(3n) ^(-n) and orthophosphate.
 9. A composition according to claim 8, wherein n is
 3. 10. A composition according to claim 1, at least 90% of said phosphate moiety being pyrophosphate.
 11. A composition according to claim 1, at least 1% of said phosphate moiety being pyrophosphate and any remaining phosphate moiety comprising phosphate of the group consisting of a ring phosphate of formula P_(n) O_(3n) ^(-n) where n is 3 and orthophosphate and combinations thereof.
 12. A composition according to claim 1, any phosphate in said phosphate moiety other than pyrophosphate being selected from the group consisting of ring phosphate of formula P_(n) O_(3n) ^(-n), one or more phosphates of formula P_(n) O.sub.(3n+1)⁻(n+2), of which not more than 15% by weight has a n value greater than 2, and combinations thereof.
 13. A sterile non-pyrogenic composition according to claim 1, wherein said stannous-phosphate complex is a freeze dried solid packaged in a sealed sterile, non-pyrogenic container purged or oxygen.
 14. A composition according to claim 13, said complex containing a pH adjusting agent to provide said stannous-phosphate complex with a pH of between 3 and 8 and being packaged in a nitrogen atmosphere in said sealed sterile, non-pyrogenic container.
 15. A sterile, pyrogen free composition for forming a bone seeking complex with technetium-99m according to claim 1, said stannous-phosphate complex being in sterile aqueous solution at a pH of between 3 and 8 for admixture with a sterile pertechnetate solution of technetium-99m to form a technetium-99m labeled stannous-phosphate complex for intravenous administration.
 16. A method of preparing a stannous-phosphate complex for use in preparing a complex thereof with technetium-99m comprising admixing a solution of a phosphate with a solid stannous salt, the phosphate moiety of said phosphate comprising pyrophosphate and containing no more than 25% by weight of linear polyphosphates of formulation P_(n) O_(3n+1) ⁻(n+2) having a molecular weight greater than pyrophosphate.
 17. A method according to claim 16, said solid stannous salt being a lyophilized stannous chloride.
 18. A method according to claim 16 said step of admixing being carried out in a non-oxidizing atmosphere.
 19. A method according to claim 18, said atmosphere being selected from a nitrogen atmosphere and a vacuum.
 20. A method according to claim 19, said phosphate solution being formed by admixing the phosphate with water under a nitrogen atmosphere and in a nitrogen flushed container, said method including the step of reducing the oxygen content of said water before forming said solution.
 21. A method according to claim 16, including the step of adjusting the pH of said stannous-phosphate complex to a pH suitable for intravenous in vivo administration into the body of a mammal and lyophilizing the resulting stannous-phosphate complex.
 22. A composition according to claim 1, wherein said stannous-phosphate complex is a freeze dried solid.
 23. A composition according to claim 22, wherein said freeze dried solid is packaged in a sealed, sterile, container.
 24. A composition for forming a complex with technetium-99m, comprising a stannous-phosphate complex, the phosphate moiety of which comprises a phosphate selected from the group consisting of (1) pyrophosphate and (2) a ring phosphate having the formula P_(n) O_(3n) ^(-n) and a molecular weight of less than 300, said phosphate moiety containing no more than 25% by weight of linear polyphosphates of formulation P_(n) O_(3n+1) ⁻(n+2) having a molecular weight greater than pyrophosphate, the weight ratio of stannous to pyrophosphate moiety ranging from 10⁻³ to 0.5.
 25. A method of preparing a stannous-phosphate complex for use in preparing a complex thereof with technetium-99m comprising admixing a solution of a phosphate with a solid stannous salt, the phosphate moiety of said phosphate being selected from the group consisting of (1) pyrophosphate and (2) a ring phosphate having the formula P_(n) O_(3n) ^(-n), said phosphate moiety containing no more than 25% by weight of linear polyphosphates of formulation P_(n) O_(3n+1) ⁻(n+2) having a molecular weight greater than pyrophosphate.
 26. A method according to claim 16 wherein the weight ratio of stannous to phosphate moiety is between 10⁻³ and 0.50.
 27. A composition for forming a complex with technetium-99m for radioactive scanning, comprising a stannous-phosphate complex, the phosphate moiety of which comprises pyrophosphate, said phosphate moiety containing no more than 25% of linear polyphosphates of formulation P_(n) O_(3n+1) ⁻(n+2) having a polyphosphate moiety of molecular weight greater than pyrophosphate, said stannous-phosphate complex being packaged in a sterile sealed container.
 28. A composition for forming a complex with technetium-99m for radioactive scanning, comprising a stannous-phosphate complex, the phosphate moiety of which comprises pyrophosphate, said phospate moiety containing no more than 25% of linear polyphosphates of formulation P_(n) O_(3n+1) ⁻(n+2) having a polyphosphate moiety of molecular weight greater than pyrophosphate, said stannous phosphate complex being in the form of a freeze dried solid.
 29. A composition for forming a complex with technetium-99m for radioactive scanning, comprising a stannous-phosphate complex, the phosphate moiety of which comprises pyrophosphate, said phospate moiety containing no more than 25% of linear polyphosphates of formulation P_(n) O_(3n+1) ⁻(n+2) having a polyphosphate moiety of molecular weight greater than pyrophosphate, said stannous-phosphate complex being in sterile aqueous solution at a pH between 3 and 8 for admixture with a sterile technetium-99m solution to form a technetium-99m labelled stannous-phosphate complex. 